Cell Mass Grade Research Articles

Trophectoderm, Inner Cell Mass, and Expansion Status for Live Birth Prediction After Frozen Blastocyst Transfer: The Winner Is Trophectoderm

Despite advancements in technologies such as time-lapse microscopy and artificial intelligence, the gold standard for embryo selection still relies on standard morphological assessment. Several studies have investigated the correlation between blastocyst characteristics (expansion status, inner cell mass, and trophectoderm) and clinical outcomes, reaching contradictory results. In consideration of these ambiguities in the literature, we performed a retrospective study of 1546 untested first-vitrified–warmed single day 5/6 blastocyst transfers. The purpose of our study is to evaluate three scenarios: (i) independent association between each morphological characteristic (expansion status, inner cell mass, and trophectoderm) and live birth; (ii) comparison between blastocysts with inner cell mass grade A and trophectoderm grade B and blastocysts with inner cell mass grade B and trophectoderm grade A; and (iii) comparison between poor-quality day 5 and top-quality day 6 blastocysts. After adjusting for principal confounders, we report that trophectoderm is more predictive of live births than inner cell mass and expansion status. We observed a trend in favor of top-quality day 6 blastocysts over poor-quality day 5 blastocysts. Moreover, on the same day of development and expansion status, blastocyst BA should be preferable to blastocyst AB.

High serum progesterone levels on the day of embryo transfer in patients undergoing artificial frozen-thawed blastocyst transfer: Is there a ceiling effect?

To evaluate the potential ceiling effect of high serum progesterone levels on the day of embryo transfer for pregnancy outcomes in patients undergoing artificial frozen-thawed blastocyst transfer (FET) cycles. This retrospective cohort study included 595 patients who underwent artificial FET cycles. We evaluated progesterone levels and found that 40.6 ng/mL corresponded to the 90th percentile and 23.9 ng/mL corresponded to the 50th percentile. Based on these findings, we categorized progesterone levels as <20 ng/mL (n=220, 37.0%), 20-40 ng/mL (n=312, 52.4%), and ≥40 ng/mL (n=63, 10.6%). The primary outcome measures were the clinical pregnancy rate (CPR) and live birth rate (LBR). Blastocyst morphology grades, including expansion, trophectoderm, and inner cell mass grades, were significantly associated with clinical pregnancy (p<0.001 for all). Progesterone levels between 20 and 40 ng/mL were associated with higher CPR (p=0.043). In the multivariate analysis, only blastocyst expansion and inner cell mass grades were independently and significantly associated with CPR [p=0.011, odds ratio (OR)=1.6, (confidence interval) CI 95%=1.13-2.39, and p=0.007, OR=1.65, CI 95%=1.14-2.39, respectively]. The progesterone level and trophectoderm grade were not statistically significant. Regarding LBR, only blastocyst expansion grades 4 and trophectoderm grades A or B were significantly associated. Based on these data, we speculate that if serum progesterone levels exceed 40 ng/mL on the day of embryo transfer in patients undergoing artificial FET cycles, there is no need to reduce the progesterone dose.

Culture media affect sex after IVF treatment-a detailed analysis of explanatory variables.

The purpose of this study was to provide a detailed analysis of clinical and laboratory factors associated with skewed secondary sex ratio (SSR) after ART. Retrospective cohort study of embryos resulting in live births, from frozen and fresh single blastocyst transfers. Embryos were cultured in either G-TL (n = 686) or Sage media (n = 685). Data was analyzed using a multivariate logistic regression model and a mixed model analysis. Significantly more male singletons were born after culture in Sage media compared to G-TL media (odds ratio (OR) 1.34, 95% CI (1.05, 1.70), P = 0.02). Inner cell mass grade B vs A (OR 1.36 95% CI (1.05, 1.76), P = 0.02) and one previous embryo transfer (OR 1.49, 95% CI (1.03, 2.16), P = 0.03) were associated with a significantly higher probability of male child at birth. Factors associated with a reduced probability of male child were expansion grade 3 vs 5 (OR 0.66, 95% CI (10.45, 0.96), P = 0.03) and trophectoderm grade B vs A (OR 0.57, 95% CI (0.44, 0.74), P = 0.00). Male embryos developed significantly faster in Sage media compared to G-TL media for the stages of blastocyst (- 1.12h, 95% CI (- 2.12, - 0.12)), expanded blastocyst (- 1.35h, 95% CI (- 2.34, - 0.35)), and hatched blastocyst (- 1.75h, 95% CI (- 2.99, - 0.52)). More male children were born after culture in Sage media compared to G-TL media. Male embryo development was affected by culture media. Our observations suggest that culture media impact male embryo quality selectively, thus potentially favoring the selection of male embryos.

Analysis of related factors influencing the detection rate of mosaic embryo and the pregnancy outcomes with mosaic embryo transfers

Objective: To explore the related factors influencing the detection rate of mosaic embryo and the pregnancy outcomes of mosaic embryo transfer in preimplantation genetic testing for aneuploidy (PGT-A) based on next generation sequencing (NGS) technology. Methods: A retrospective study was performed to analyze the clinical data of patients in 745 PGT-A cycles from January 2019 to May 2023 at Chongqing Health Center for Women and Children, including 2 850 blastocysts. The biopsy cells were tested using NGS technology, and the embryos were divided into three groups based on the test results, namely euploid embryos, aneuploid embryos and mosaic embryos. The influence of population characteristics and laboratory-related parameters on the detection rate of mosaic embryo were analyzed, and the pregnancy outcomes of 98 mosaic embryo transfer cycles and 486 euploid embryo transfer cycles were compared during the same period, including clinical pregnancy rate and live birth rate. Results: Among the embryos tested (n=2 850), the number and proportion of euploid embryos, aneuploid embryos and mosaic embryos were 1 489 (52.2%, 1 489/2 850), 917 (32.2%, 917/2 850) and 444 (15.6%, 444/2 850), respectively. Among mosaic embryos, 245 (55.2%, 245/444) were segmental mosaic embryos, 118 (26.6%, 118/444) were whole-chromosome mosaic embryos, and 81 (18.2%, 81/444) were complex mosaic embryos. NGS technology was performed in 4 genetic testing institutions and the detection rate of mosaic embryo fluctuated from 13.5% to 27.0%. The distributions of female age, level of anti-Müllerian hormone, PGT-A indications, ovulation-inducing treatments, gonadotropin (Gn) dosage, Gn days, inner cell mass grade, trophectoderm cell grade, genetic testing institutions and developmental stage of blastocyst were significantly different among the three groups (all P<0.05). Multi-factor analysis showed that the trophectoderm cell grade and genetic testing institutions were significantly related to the detection rate of mosaic embryo; compared with the trophectoderm cell graded as A, the detection rate of mosaic embryo was significantly increased in the trophectoderm cell graded as B-(OR=1.59, 95%CI: 1.04-2.44, P=0.033); compared with genetic testing institution a, the detection rate of mosaic embryo was significantly higher (OR=2.89, 95%CI: 2.10-3.98, P<0.001) in the testing institution c. The clinical pregnancy rate and live birth rate with mosaic embryos transfer were significantly lower than those of euploid embryos transfer (clinical pregnancy rate: 51.0% vs 65.2%, P=0.008; live birth rate: 39.4% vs 53.2%, P=0.017). After adjustment for age, PGT-A indications, trophectoderm cell grade and days of embryo culture in vitro, the clinical pregnancy rate and live birth rate with mosaic embryos transfer were significantly lower than those of euploid embryos transfer (clinical pregnancy rate: OR=0.52, 95%CI: 0.32-0.83, P=0.007; live birth rate: OR=0.50, 95%CI: 0.31-0.83, P=0.007). Conclusions: The trophectoderm cell grade and genetic testing institutions are related to the detection rate of mosaic embryo. Compared with euploid embryos transfer, the clinical pregnancy rate and live birth rate with mosaic embryos transfer are significantly reduced. For infertile couple without euploid embryos, transplantable mosaic embryos could be recommended according to the mosaic ratio and mosaic type in genetic counseling to obtain the optimal pregnancy outcome.

P-208 Culture media affect gender after IVF treatment: A detailed analysis of explanatory variables

Abstract Study question Does embryo culture media affect gender at birth and why? Summary answer More male children were born after culture in the Sage 1-Step media compared to the G-TL media. What is known already ART increases the proportion of male children at birth. Laboratory techniques and culture conditions is known to impact secondary sex ratio (SSR). Physiological development differs in male and female embryos, leading to different metabolic requirements at the preimplantation stage. The composition of commercial culture medias varies and thus, the different medias may have different impact on embryo development and quality according to gender. This, in turn, may influence SSR, as embryos are selected based on morphological scoring systems and developmental speed. Study design, size, duration The study was designed as a retrospective registry-based study with all data collected at a single clinic. All embryos resulting in live birth from frozen and fresh single blastocyst transfers, in the period from 1. January 2017 to 31. December 2020 were included. Donor embryos and embryos cultured in more than one culture media were excluded. A total of 1371 SET embryos were included. Participants/materials, setting, methods Embryos were cultured in either the G-TL media, Vitrolife (n = 686) or the Sage 1-Step media, Origio (n = 685). All embryos were monitored for 5-6 days in a time-lapse incubator, Vitrolife, before single blastocyst transfer. Multivariate logistic analysis of cycle and embryo characteristics was performed to identify all factors associated to sex. The association between culture media and embryo morphokinetic according to sex was evaluated using a mixed model analysis. Main results and the role of chance Significantly more male singletons were born after culture in 1-Step media compared to G-TL media (risk ratio (RR) 1,1 95% CI [1,0, 1,3], P = 0,01). The female/male ratio was 42,3/57,7% in 1-Step media and 48,5/51,5% in G-TL media. The multivariate regression analysis displayed a higher chance of a male child with higher expansion grade (grade 5) (RR, 1,2 95% CI [1,0, 1,4] P = 0,04), lower inner cell mass grade (grade B) (RR 1,13 95% CI [1,0, 1,3] P = 0,02) and second embryo transfer (RR 1,2 95% CI [1,0, 1,3] P = 0,03). Trophectoderm (TE) grade B reduced the probability of male child compared with TE grade A (RR 0,8 95% CI [0,8, 0,9] P &amp;gt; 0,05). Male embryos developed significantly faster in the 1-Step media compared to the G-TL media for the stages of; blastocyst (tB) (-1,1 hours 95% CI [-2,1, -0,1]), expanded blastocyst (tEB) (-1,3 hours 95% CI [-2,3, -0,3]) and hatched blastocyst (tH) (-1,7 hours 95% CI [-3,0, -0,5]). Timing of development were the same for female embryos in the G-TL media compared to the 1-Step media. There was no significant difference in timing of development between female and male embryos in identical culture media. Limitations, reasons for caution This study was a retrospective study with data collection from only a single clinic, and as such in risk of confounding. Apart from culture media, culture conditions were similar during the two time periods. Paternal factors and type of infertility was not included. Wider implications of the findings Our observations suggest that culture media impact male embryo quality selectively, thus favoring selection of male embryos. As the impact appears to vary between different culture media, and has a measurable impact on gender after birth, the influence of culture media on embryo quality deserves more attention. Trial registration number not applicable

Day-3-embryo fragmentation is associated with singleton birth weight following fresh single blastocyst transfer: A retrospective study.

BackgroundPrevious studies have arguably associated poor embryo morphology with low birth weight in singletons following single embryo transfer. However, the association between birth weight and specific morphological features in the cleavage stage remains less known. The purpose of the study was to investigate whether morphological features of embryos at the cleavage stage affect birth weight following blastocyst transfer.MethodsThe single-center retrospective cohort study included 4,226 singletons derived from fresh single cleavage-stage embryo transfer (ET; n = 1,185), fresh single blastocyst transfer (BT; n = 787), or frozen-thawed single blastocyst transfer (FBT; n = 2,254) between 2016 and 2019. Morphological parameters including early cleavage, day-3 fragmentation, symmetry, blastomere number, and blastocyst morphology were associated with neonatal birth weight and birth weight z-score in multivariate regression models. The models were adjusted for maternal age, body mass index (BMI), parity, peak estradiol level, endometrial thickness, insemination protocol, female etiologies, order of transfer, mode of delivery, and year of treatment.ResultsAdjusted for confounders, day-3 fragmentation was the only morphological feature associated with birth weight and birth weight z-score, while early cleavage, symmetry, blastomere number, and blastocyst morphology were not. Day-3 fragmentation increased the birth weight in both the ET (115.4 g, 95% CI: 26.6–204.2) and BT groups (168.8 g, 95% CI: 48.8–288.8) but not in the FBT group (7.47 g, 95% CI: -46.4 to 61.3). The associations between birth weight and these morphological parameters were confirmed through birth weight z-score analyses. The adjusted odds of large for gestational age (LGA) and high birth weight were also significantly greater in singletons following the transfer of fragmented embryos in the BT group [odds ratio (OR) 3, 95% CI: 1.2–7.51 and OR 3.65, 95% CI: 1.33–10, respectively]. The presence of fragmentation at the cleavage stage also affected the association between the blastocyst morphology and birth weight. Inner cell mass grades were negatively associated with birth weight in blastocysts with day-3 fragmentation but not in blastocysts without.ConclusionsThe birth weight following blastocyst transfer was found to be positively associated with fragmentation at the cleavage stage. The data did not support the argument that transferring a poor-looking embryo may increase the risks of low birth weight. However, concerns for LGA infants remain.

P-683 High Serum Progesterone Levels on Day of Embryo Transfer for Pregnancy Outcomes in Patients Undergoing Artificial Frozen-thawed Blastocyst Transfer; Is There a Ceiling Effect?

Abstract Study question Is there a ceiling effect of high serum progesterone levels on day of embryo transfer for pregnancy outcomes in patients undergoing artificial frozen-thawed blastocyst transfer? Summary answer There is no ceiling effect of high serum progesterone levels on day of embryo transfer(&amp;gt;40 ng/mL)for pregnancy outcomes in patients undergoing artificial frozen-thawed blastocyst transfer. What is known already In recent studies, 10 ng/mL was usually used as the lower threshold value for serum progesterone (P4) level on the day of embryo transfer in patients undergoing artificial frozen-thawed blastocyst transfers and pregnancy outcomes were compared between patients with P4 levels above and below this threshold. The main reason for choosing this threshold in the studies is that it reflects the healthy corpus luteum function. Although many studies have investigated the lower threshold value, there is paucity of data on the high threshold value that can indicate the possible ceiling effect of P4 in the literature. Study design, size, duration This was a retrospective cohort study including 595 patients undergoing artificial frozen-thawed blastocyst transfer between 2017 to 2021. In order to eliminate the age-related bias, only patients performed euploid embryo transfer according to preimplantation genetic testing for aneuploidy from patients above aged 35 years were included. Participants/materials, setting, methods When we evaluated the percentiles of according to the progesterone levels,40.6 ng/ml corresponded to 90th percentile and 23.9 ng/ml corresponded to 50th percentile. Based on this finding, rounding up the numbers, we determined the progesterone levels cut-off as &amp;lt; 20ng/mL,n=220(37.0%);20-40ng/mL,n=312(52.4%) and ≥40ng/mL,n=63(10.6%). The main outcome measure was pregnancy outcomes as clinical pregnancy and live birth rate. We compared pregnancy outcomes according to age, BMI, blastocyst expansion, trophectoderm and inner cell mass grade and progesterone cut-off levels . Main results and the role of chance The median age of the patients was 31 (Range 20-46) and the mean body mass index (BMI) of the patients was 24.5±4.0 kg/m2. The overall clinical pregnancy rate and live birth rate were 61.8% (368/595) and 52.9% (315/595), respectively. Patients were subdivided into two different groups according to clinical pregnancy and live birth rate outcomes as follows; positive and negative. Age and BMI of patients were comparable between groups. Blastocyst morphology grade as expansion, trophectoderm and inner cell mass grade are statistically significantly associated with clinical pregnancy (p&amp;lt;.001 for all) and progesterone level between 20-40ng/mL is associated with higher clinical pregnancy rate (p:0.043). In multivariate analysis; only blastocyst expansion and inner cell mass grade were the independently and significantly factors that are associated with clinical pregnancy rate (p:0.011, OR: 1.6, CI95%: 1.13-2.39 and p:0.007, OR: 1.65, CI95%: 1.14-2.39, respectively). Progesterone level and trophectoderm grade were not fount statistically significant (p: 0.310 and p:0.489, respectively). In evaluation of the association between factors and live birth rate, only blastocyst expansion grade equal or over 4 and trophectoderm grade A or B were associated with statistically significant live birth rates. Limitations, reasons for caution The main limitations of current study were the retrospective design and the limited sample size of the ≥40 ng/mL cohort of serum progesterone levels. Wider implications of the findings According to this data, we speculated that if the serum P4 level is above 40 ng/mL on the day of embryo transfer in patients undergoing artificial frozen-thawed blastocyst transfer, there is no need to reduce the dose of progesterone used. Trial registration number N/A

P-146 Blastocyst morphometry and morphology predict ploidy

Abstract Study question Are objective measurements and subjective assessments of blastocysts predictive of aneuploidy? Summary answer Aneuploidy was predicted equally well by lower trophectoderm cell count (objectively measured morphometry) or by a lower subjective trophectoderm grade. What is known already Blastocyst morphological grade has been reported to be moderately predictive of embryo ploidy. However, due to its subjective nature, morphological grade may be significantly more difficult to code into artificial intelligence algorithms when compared to objective measurements. Study design, size, duration This retrospective cohort study included all 1409 blastocysts that were subject to pre-implantation genetic testing (PGT) in a 30-month study period. Participants/materials, setting, methods Per clinic routine, embryos from oocyte were cultured to the blastocyst stage following conventional ovarian stimulation, oocyte retrieval, and intracytoplasmic sperm injection. If the patients opted for PGT, a laser was used to open the zona at the cleavage stage, trophectoderm biopsies were collected at the blastocyst stage, and biopsies were analyzed by next-generation sequencing. Inner cell mass sizes were measured by ocular micrometer and trophectoderm cells were counted in one plane of focus. Main results and the role of chance The mean patient age (including oocyte donors) was 31.9±6.4 years and the mean number of collected oocytes was 23.0±12.9. In multivariable logistic regression, greater patient age was the best available predictor of blastocyst aneuploidy (P &amp;lt; 0.0001). Along with age in the model, the observation of fewer trophectoderm cells (P = 0.0019) was also predictive of aneuploidy. Alternatively, the failure to have an A-grade trophectoderm (P = 0.0007) was also predictive of aneuploidy, also with age in the model. Neither the objectively measured inner cell mass size nor subjective inner cell mass grade were significant predictors of ploidy. Overall, objectively measured blastocyst morphometry was about equally predictive of ploidy as was subjective blastocyst grading, and area under the receiver operating characteristic curve was 0.62 for each model. Limitations, reasons for caution This study was retrospective, allowing the possibility of selection bias among patients and among embryos chosen for biopsy. Wider implications of the findings Artificial intelligence algorithms assessing embryos might benefit from the similar performance of subjective grading and objective measurements, because it is much easier to code objective measurements into the algorithms. Trial registration number None.

P-402 The association between embryo morphology and first trimester miscarriage after single blastocyst transfer

Abstract Study question Can standard morphological assessment of blastocyst quality accurately predict first trimester miscarriage? Summary answer Decreasing trophectoderm and inner cell mass grade equally predicted first trimester miscarriage independently. After adjusting for confounders, poor embryo quality was not associated with miscarriage. What is known already Embryo quality determined by morphological assessment is a well-established predictor of successful implantation and live birth with in vitro fertilization treatment. Chromosomal abnormalities account for over half of first trimester miscarriages, and links between ploidy status and embryo morphology have been demonstrated. However, the association between blastocyst morphology and first trimester miscarriage is still poorly understood with consensus opinion yet to be established. Study design, size, duration This single centre retrospective cohort study included 2020 fresh and frozen single embryo transfers between October 2010 and October 2020. Blastocyst degree of expansion, inner cell mass (ICM) grade, and trophectoderm (TE) grade were compared for 354 pregnancies ending in first trimester miscarriage and 1666 pregnancies that surpassed 12 weeks of gestation. The association between blastocyst morphology and miscarriage was examined by logistic regression analysis. Participants/materials, setting, methods All single blastocyst transfers over a 10-year period resulting in first trimester miscarriage or pregnancy beyond 12 weeks of gestation were included in our analysis. Cycles involving donor oocytes, preimplantation genetic testing, resulting in biochemical pregnancy, or ectopic pregnancy were excluded. Differences between groups were analysed with chi-square and logistic regression analysis. Odds ratios (aOR) for miscarriage versus ongoing pregnancy were adjusted for maternal age, cycle type, blastocyst expansion, ICM grade, and TE grade. Main results and the role of chance Out of 2020 single embryo transfer cycles, a total of 354 resulted in first trimester miscarriage (17.5%). Independently, miscarriage rates increased with decreasing ICM grade (A to C; 14.6%, 18.9%, and 26.5%, p = 0.003) and TE grade (A to C; 14.6%, 17.7%, and 26.6%, p = 0.004). Univariate logistic regression showed higher risks of miscarriage with ICM grade C compared to ICM grade A (OR 2.241, 95%CI 1.292-3.886, p = 0.004), TE grade C compared to TE grade A (OR 2.116, 95%CI 1.360 – 3.292, p = 0.001), and expansion grades of 1 and 2 compared to expansion of 5 and 6 (OR 2.514, 95%CI 1.477 – 4.278, p = 0.001). After multivariable logistic regression adjusting for maternal age at embryo transfer, cycle type, and all morphological parameters (degree of expansion, ICM, and TE), only expansion grade of 1 and 2 showed a statistically significant association with first trimester miscarriage. Limitations, reasons for caution The main limitation of this study is the retrospective design of the analysis, and subjective nature of embryo morphological assessment. There may also be residual confounding by unknown or unmeasured confounders such as paternal age or maternal BMI. Wider implications of the findings Embryo selection for transfer should continue to be guided by standard morphological assessment. Decreasing grade of ICM and TE are both positively associated with an increased miscarriage risk, while poorly expanded blastocysts are most predictive of first trimester miscarriage. Patients undergoing embryo transfer should be adequately counselled about these risks. Trial registration number Not applicable

The Relationship between Human Embryo Parameters and De Novo Chromosomal Abnormalities in Preimplantation Genetic Testing Cycles.

Design In total, 456 PGT cycles, including 283 PGT-SR cycles and 173 PGT-A cycles, were assessed through comprehensive chromosome screening (CCS) from January 2017 to June 2020 at the Department of Reproductive Medicine of the Third Affiliated Hospital of Zhengzhou University. Trophectoderm (TE) biopsies were sequenced using next-generation sequencing (NGS). The incidence of de novo chromosome abnormalities was calculated, and the relationships between de novo chromosome abnormality rates and maternal age, number of oocytes retrieved, and parameters of cleavage-stage embryos and blastocyst-stage embryos were investigated. Results The incidence of de novo chromosome abnormalities was 28.0% (318/1,135) in the PGT-SR cycles and 36.3% (214/590) in the PGT-A cycles, which increased with maternal age in both PGT-SR cycles (P = 0.018) and PGT-A cycles (P < 0.001). The incidence of de novo chromosome abnormalities was related to TE grade (P < 0.001), internal cell mass grade (P = 0.002), and development speed (day 5 vs. day 7: P < 0.001) of blastocyst-stage embryos. The incidence of de novo chromosomal abnormalities was irrelevant to the number of oocytes retrieved and the parameters of the embryo at the cleavage stage. Conclusion Blastocysts with higher morphology scores and faster progression had a lower incidence of de novo chromosome abnormalities, especially complex chromosome abnormalities. De novo chromosome abnormalities may negatively affect the morphological grading of blastocysts. Our findings will provide valuable information to the fertility doctor for embryo selection in non-PGT cycles.

Evaluation of clinical parameters as predictors of monozygotic twins after single frozen embryo transfer

ObjectiveTo determine if recent evolutions in laboratory protocols, including the increased use of natural cycles and the use of a hyaluronan-containing transfer medium, affected the rate of monozygotic twin (MZT) pregnancies after single frozen embryo transfer (FET).DesignRetrospective cohort study.SettingUrban university-based fertility center.Patient(s)Patients who underwent single FET between January 2016 and December 2018 resulting in an intrauterine pregnancy.Intervention(s)Transition to a transfer protocol with a hyaluronan-containing transfer medium in July 2017.Main Outcome Measure(s)Number of MZT pregnancies.Result(s)There were 1,619 cycles that met the inclusion criteria and 31 (1.9%) resulted in MZT pregnancies. A hyaluronan-containing transfer medium was used in 875 (54.1%) cycles. Programmed cycles were used for 1,385 (85.5%) FETs and 234 (14.5%) cycles were natural. The mean age at FET, oocyte age, endometrial echo thickness, inner cell mass grade, trophectoderm grade, expansion, and day of blastocyst vitrification were similar between the groups. The use of a hyaluronan-containing transfer medium resulted in fewer MZTs. After controlling potential confounders with a multivariate regression, the use of the hyaluronan-containing medium still resulted in fewer MZTs. Monozygotic twins were colinear with preimplantation genetic testing (PGT), so PGT was excluded as a variable in our regression. A regression of PGT only cycles showed that the use of the hyaluronan-containing medium was still associated with a reduction in MZT pregnancies.Conclusion(s)The use of a hyaluronan-containing transfer medium was associated with a lower rate of MZTs. Other clinical parameters, including cycle type, were not associated with changes in the number of MZTs. The use of PGT needs to be further investigated as a risk factor for MZTs.

Monochorionic twins after single blastocyst transfer: retrospective cohort and blinded time lapse annotation analysis

Research questionIs it possible to identify monochorionic twin pregnancies before blastocyst transfer based on maternal or embryo characteristics registered by time lapse? DesignA retrospective cohort study including women who received fertility treatment (n = 6501) between 2010 and 2019 at two fertility clinics in central Denmark. The treatment resulted in 2239 pregnancies after single embryo transfer (SET) and, of those, 43 (1.92%) were monochorionic twins. Baseline characteristics, information on assisted reproductive technology and ultrasonic findings at gestational week 8 were collected on all women. Furthermore, a blinded time lapse annotation analysis was conducted by two independent laboratory technicians on a total of 85 embryos. A total of 22 embryos leading to monochorionic pregnancies were matched with 63 embryos leading to singleton pregnancies. ResultsA monochorionic twin rate of 1.92% was found. No significant difference was found in maternal age, the use of intracytoplasmic sperm injection compared with IVF, indication for treatment or other maternal characteristics. In the blinded annotation analysis, inner cell mass grade A was associated with an increased risk of twinning (P = 0.04) in fresh embryos. The s3 division timing was found to be significantly shorter in fresh twin compared with singleton embryos (P = 0.006). No other time lapse parameters were found to be characteristic of twin embryos. ConclusionTo the best of our knowledge, this is the first blinded annotation study to identify aspects in time lapse resulting in monochorionic twins after SET. Whether inner cell mass grade A and s3 reflect a biological background for monochorionic twinning merits further investigation.

Prediction of embryo survival and live birth rates after cryotransfers of vitrified blastocysts

Research questionWhich pre-vitrification parameters are the most predictive of survival and live birth in vitrified–warmed blastocyst transfer cycles? DesignA retrospective study including 11,936 warmed blastocysts. Pre-vitrification morphological parameters analysed for blastocysts included day of vitrification; blastocyst expansion degree; trophoectoderm grade (A, B and C); and inner cell mass grade (A, B and C). Univariate and multivariate generalized estimating equations models were used to analyse survival, clinical pregnancy and live birth rate. A stepwise regression analysis was conducted to select and classify by order which outcomes were the most predictive. ResultsThe odds of survival increased almost twice for blastocysts with lower expansion degree (OR 1.92; 95% CI 1.37 to 2.69; P < 0.001) and by about 50% for blastocysts vitrified on day 5 (OR 1.56; 95% CI 1.27 to 1.89; P < 0.001). Multivariate generalized estimating equations model showed that trophectoderm grade followed by the day of vitrification were the most significant predictors of live birth. The odds of live birth increased nearly three times for blastocysts with trophectoderm graded as A compared with those with trophectoderm graded as C (OR 2.85; 95% CI 2.48 to 3.27; P < 0.001), and double for blastocysts vitrified on day 5 compared with those vitrified on day 6 (OR 2.22; 95% CI 1.97 to 2.49; P < 0.001). The odds of live birth also increased in higher expansion degree blastocysts. ConclusionsBlastocysts vitrified on day 5 and those with higher trophoectoderm grade should be given priority when warming.

Association between blastocyst morphology and live birth rate following frozen-thawed single blastocyst transfer: Results from a 5-year retrospective analysis of 2593 cryopreserved blastocysts.

Our aim was to investigate associations between blastocyst morphology parameters and live birth outcome and to make possible additional recommendations for existing embryo selection strategies. This retrospective cohort study included 2593 frozen-thawed single blastocyst transfers (SBT) cycles from 2012 to 2016. Multivariable logistic regression model was used to analyze the independent predictive effectiveness of blastocyst parameters for live birth rate (LBR). The participants enrolled in the present study were 32 (28-35) years old with a median body mass index of 21.20 (19.60-23.40) kg/m2 , among whom 1058 (40.8%) women had live births. Among the three blastocyst morphology parameters, we found only inner cell mass grade and trophectoderm cell grade had significant effects on LBR (P < 0.001). When adjusting for potential confounders in a multivariable logistic regression model, the expansion and hatching (EH) stage of blastocoel also showed obvious correlation with LBR. Blastocysts at EH stage 4-5 had a significantly higher LBR than that at stage 3 (P < 0.05). Additionally, the timing of blastulation was also an important predictor of LBR. Blastocysts vitrified on day 6 and day 7 yielded a lower LBR than that vitrified on day 5 (P < 0.001). The timing of blastulation and all blastocyst morphology parameters were associated with LBR independently. Although the most important parameter for predicting clinical outcomes remains undetermined, the timing of blastulation was a stable predictor of live birth for frozen-thawed SBT.

Overall Blastocyst Quality, Trophectoderm Grade, and Inner Cell Mass Grade Predict Pregnancy Outcome in Euploid Blastocyst Transfer Cycles.

Background:Despite recent advances that have improved the pregnancy success rates that can be achieved via in vitro fertilization (IVF) therapy, it is not yet clear which blastocyst morphological parameters best predict the outcomes of single blastocyst transfer. In addition, most of the previous studies did not exclude the effect of embryo aneuploidy on blastocysts transfer. Thus, the present study investigated the predictive value of various parameters on the pregnancy outcomes achieved via the transfer of frozen euploid blastocysts.Methods:The study retrospectively analyzed 914 single euploid blastocyst transfer cycles that were performed at the Peking University Third Hospital Reproductive Medical Center between June 2011 and May 2016. The expansion, trophectoderm (TE), and inner cell mass (ICM) quality of the blastocysts were assessed based on blastocyst parameters, and used to differentiate between “excellent”, “good”, “average”, and “poor”-quality embryos. The relationship between these embryo grades and the achieved pregnancy outcomes was then analyzed via the Chi-square and logistic regression tests.Results:For embryo grades of excellent, good, average and poor, the clinical pregnancy rates were 65.0%, 59.3%, 50.3% and 33.3%, respectively; and the live-birth rates were 50.0%, 49.7%, 42.3% and 25.0%, respectively. Both the clinical pregnancy rate (χ2 = 21.28, P = 0.001) and live-birth rate (χ2 = 13.50, P < 0.001) increased with the overall blastocyst grade. Both rates were significantly higher after the transfer of a blastocyst that exhibited either an A-grade OR B-grade TE, and similarly, an A-grade ICM, than after the transfer of a blastocyst that exhibited a C-grade TE and/or ICM. The degree of blastocyst expansion had no apparent effect on the clinical pregnancy or live-birth rate. All odds ratio were adjusted for patient age, body mass index, length (years) of infertility history, and infertility type.Conclusions:A higher overall euploid blastocyst quality is shown to correlate most strongly with optimal pregnancy outcomes. The study thus supports the use of the described TE and ICM morphological grades to augment current embryo selection criteria.

Morphologic grading of euploid blastocysts influences implantation and ongoing pregnancy rates

To determine whether blastocyst grading can predict pregnancy outcomes in the frozen-thawed embryo transfer (FET) of euploid blastocysts. Retrospective cohort study. Academic medical center. Women who underwent FET of euploid embryo(s) between January 2013 and December 2015, with blastocysts were divided into four groups based on their morphologic grading before cryopreservation: excellent (≥3AA), good (3-6AB, 3-6BA, 1-2AA), average (3-6BB, 3-6AC, 3-6CA, 1-2AB, 1-2BA), and poor (1-6BC, 1-6CB, 1-6CC, 1-2BB). FET. Ongoing pregnancy rate (OPR). A total of 417 FET cycles (477 embryos) were included. Excellent-quality embryos (n = 38) yielded a statistically significantly higher OPR than poor-quality embryos (n = 106) (84.2% vs. 35.8%; adjusted odds ratio 11.0; 95% confidence interval, 3.8-32.1) and average-quality embryos (n = 197) (84.2% vs. 55.8%; adjusted odds ratio 4.8; 95% confidence interval, 1.7-13.3). Good-quality embryos (n = 76) were associated with a statistically significantly higher OPR than poor-quality embryos (61.8% vs. 35.8%). These odds ratios were adjusted for patient's age, body mass index, number of transferred embryos, type of frozen cycle, peak endometrial thickness, day of trophectoderm biopsy (5 or 6), and total number of euploid embryos for each patient. An inner cell mass grade of A yielded a statistically significantly higher OPR than ICM grade C (76.2% vs. 13.5%) or grade B (76.2% vs. 53.6%) after controlling for all confounders. Contrary to prior published studies, the current data suggest that blastocyst morphologic grading and particularly inner cell mass grade is a useful predictor of OPR per euploid embryo. Morphologic grading should be used to help in the selection among euploid blastocysts.

Birth weight is associated with inner cell mass grade of blastocysts

To determine the relationship between blastocyst growth parameters and birth weight. Cohort study. University-affiliated fertility center. In vitro patients who delivered a singleton after a single-blastocyst transfer. None. Birth weight adjusted for gestational age at delivery and gender, with adjusted birth weight examined for association with blastocyst scores and grades. After standard in vitro fertilization (IVF) and thawed embryo transfers, greater birth weight was associated with a higher inner cell mass grade. The grade of the trophectoderm and stage of the blastocyst did not relate to weight. Embryonic growth as early as day 5 can predict the progress of fetal development as measured by birth weight.

Blastocyst expansion and inner cell mass grades are associated with clinical pregnancy in fresh single transfers

Various recent studies have pointed out in the direction of the trophectoderm as one of the most reliable predictors of pregnancy but so far there is no scientific consensus about which morphological blastocyst parameter weighs more in the selection of the embryo with the greatest chance of success. We designed a study to further investigate the individual contribution of each parameter to clinical pregnancy.

Association between blastocyst morphology and outcome of single-blastocyst transfer

The aim of this study was to assess the ability of three individual blastocyst morphology parameters – expansion and hatching (EH) stage, inner cell mass (ICM) grade and trophectoderm grade – to predict outcome of a cycle with single-blastocyst transfer. The study was a secondary analysis of data prospectively collected in a large multicentre trial. A total of 618 intracytoplasmic sperm injection patients undergoing ovarian stimulation in a gonadotrophin-releasing hormone antagonist cycle with compulsory single-blastocyst transfer on day 5 were included. In the simple logistic regression analysis, all three blastocyst morphology parameters were statistically significantly (P<0.005 for each) associated with positive human chorionic gonadotrophin, clinical and ongoing pregnancy rates and live birth rates, while only the ICM grade was significantly (P=0.033) associated with early pregnancy loss rate. Blastocyst EH stage was the only significant predictor of live birth (P=0.002) in the multiple logistic regression. In conclusion, although all three blastocyst morphology parameters were related to treatment outcome of fresh single-blastocyst cycles, selection of high-quality blastocysts for transfer should consider first the EH stage. Transfer of a blastocyst with ICM grade A may reduce the risk of early pregnancy loss.Choosing the embryo(s) with the best implantation potential is essential for securing each couple the highest chance of achieving pregnancy after assisted reproduction. The selection of embryo(s) for transfer at the blastocyst stage is based on morphology parameters of expansion and hatching stage, inner cell mass grade and trophectoderm grade. The aim of this study was to assess the relative impact of each parameter in predicting the probability of a successful outcome. The study was a secondary analysis of data prospectively collected in a large multicentre trial. A total of 618 patients who underwent single-blastocyst transfer on day 5 were included. Statistical analysis showed that all three blastocyst morphology parameters were significantly associated with positive human chorionic gonadotrophin (βHCG), clinical and ongoing pregnancy rates and live birth rates. Only the inner cell mass grade was significantly associated with early pregnancy loss between the positive βHCG test and confirmation of ongoing pregnancy 10–11weeks after transfer. The expansion and hatching stage was the only significant predictor of live birth in the multiple logistic regression analysis. In conclusion, although all three blastocyst morphology parameters were related to treatment outcome of fresh single-blastocyst cycles, selection of high-quality blastocysts for transfer should consider first the expansion and hatching stage. Transfer of a blastocyst with inner cell mass grade A may reduce the risk of early pregnancy loss.

Trophectoderm grade, but not inner cell mass grade, is predictive of outcomes in single blastocyst transfers

Trophectoderm grade, but not inner cell mass grade, is predictive of outcomes in single blastocyst transfers